PROJECT SUMMARY Intestinal epithelial cells (IECs) that reside at the interface between the microbiota and diverse immune cell populations are critical for initiating and maintaining However, the mechanisms underlying how IECs tissue-intrinsic defenses needed to quickly combat infection. orchestrate the dynamic cross-talk between microbiota, epithelial cells, and lymphocytes remain poorly defined. Epigenetic-modifying enzymes represent a powerful, but poorly understood, interface by which mammalian cells can respond to microbial signals and regulate host response. We recently identified that IEC deletion of the epigenetic regulator HDAC3 in utero inhibited microbiota-dependent regulation of IECs and impaired activation of intestinal immune cells during infection. However, it remains unknown how HDAC3 in IECs actively governs intestinal immunity or sustains microbiota- sensitive defense against enteric infection. The goals of this proposal are to interrogate how differentiated and progenitor IECs actively control intestinal immune cell dynamics and microbiota-sensitive pathways needed for antibacterial immunity through epigenetic regulation. Based on new preliminary findings, we hypothesize that HDAC3 is an essential epithelial factor that (1) dynamically coordinates resident lymphocytes in the intestine and (2) primes short- and long-term IEC responsiveness to infection by integrating signals from the microbiota. Employing Citrobacter rodentium, a murine model of pathogenic human Escherichia coli infection, along with an exciting array of inducible transgenic mouse tools, germ-free mice, and human organoids, three specific aims are proposed to investigate these hypotheses. We will (i) interrogate how active regulation of IECs by HDAC3 directs tissue-resident lymphocyte dynamics and test how this epithelial-immune cell relationship is mechanistically controlled, (ii) directly examine how the microbiota promote epithelial antimicrobial secretion during infection, and (iii) employ reporter mice to define whether epigenetic regulation of stem cells mediates how the microbiota sustains long-term defense in the intestine. We will also translate our murine studies to human intestinal organoids to provide mechanistic insights on the role of HDAC3 and epigenetics in host-microbe interactions in human intestine. This work will uncover novel mechanisms for how IECs integrate microbial signals to instruct intestine-intrinsic immunity and guide design of next generation therapeutics that can epigenetically prime the intestine to effectively defend against enteric infections.